(i) Field of the Invention
The present invention relates to a process for increasing the phosphate content of starches of genetically modified plant cells in comparison with starches from corresponding genetically nonmodified wild-type plant cells, wherein a plant cell is genetically modified by the introduction of a foreign nucleic acid molecule which codes for a soluble starch synthase II, and this starch synthase II is overexpressed.
Furthermore, the present invention relates to rice starch and rice flour with improved quality properties, to rice grains comprising this rice starch, and the rice plants on which these rice grains grow.
(ii) Description of the Related Art
Rice is the most important food for more than half of the world's population. In some countries, rice amounts to approximately 80% of all the food intake. The annual production worldwide is 550 million tonnes of rice.
The rice kernel consists of approximately 76% of starch and approximately 7-8% of protein. It contains only 1.3% of fat and a large number of trace elements (0.6%) such as phosphorus, iron and magnesium.
The economically most important rice species is Oryza sativa, whose basic varieties can be divided into two groups:    the “indica” group, which includes only long-grain rice, and    the “japonica” group, which contains medium- and short-grain rice.
Long-grain rice (rice varieties whose grains are separate when cooked) comes mainly from India or Java; short-grain rice (such as “pudding rice”, i.e. rice varieties whose grains are sticky when cooked) come primarily from Japan. The varieties from China and South East Asia are halfway between the above.
In all varieties, in turn, there are two main types: translucent grain or opaque grain. These differ in the composition of their starch: the starch of translucent rice consists of approximately 20% of amylose and to 80% of amylopectin, while that of the opaque rice, in contrast, consists virtually only of amylopectin.
Amylopectin has a specific cluster structure and is synthesized by a variety of subunits or isoforms of four classes of enzymes: soluble starch synthase (SS), starch-branching enzymes (SBE), starch-debranching enzymes (SDE) and ADP glucose pyrophosphorylase (Nakamura 2002, Plant Cell Physiol. 43(7): 718-725).
The cooking and eating characteristics are determined mostly by the amylose content of the rice endosperm. Varieties with a low amylose content are damp and sticky after cooking, while grains with a high amylose content go dry and fluffy upon cooking (H. ten Have in Hoffmann Mudra Plarre (HMP) 1985: Lehrbuch der Züchtung landw. Kulturpflanzen, Volume 2, pp. 110-123).
Grain quality is very important not only for the consumer, but also for the milling industry; grain properties such as grain size, grain shape and grain quality are important features since they affect the yield of ground rice and the percentage of broken grains (ten Have 1985, supra).
Rice flour is relatively neutral in taste and therefore very suitable as the basis for mild-tasting products or else as an admixture. Owing to its hypoallerginicity, it is also very suitable for baby formula or as diet for allergy sufferers.
Besides oils, fats and proteins, polysaccharides are the most important renewable raw materials from plants. Besides cellulose, starch, which is one of the most important storage materials in Higher Plants, is of prime importance among the polysaccharides.
The polysaccharide starch is a polymer of chemically uniform units, the glucose molecules. However, it constitutes a highly complex mixture of different forms of molecules which differ with regard to their degree of polymerization and the occurrence of branchings of the glucose chains and their chain length and, additionally, may be derivatized, for example phosphorylated. Starch therefore does not constitute a uniform raw material. In particular, amylose starch, an essentially unbranched polymer of α-1,4-glycosidically linked glucose molecules, differs from amylopectin starch, which, in turn, is a complex mixture of differently branched glucose chains. The branchings are formed by the occurrence of additional α-1,6-glycosidic linkages. In typical plants used for industrial starch production, for example maize, wheat or potato, the starch synthesized is approximately 20%-25% amylose starch and approximately 70%-75% amylopectin starch.
The functional properties of starch are affected greatly not only by the amylose/amylopectin ratio and the phosphate content, but also by the molecular weight, the pattern of the side-chain distribution, the ionic content, the lipid and protein content, the mean size of the starch grains, the starch grain morphology and the like. Important functional properties which may be mentioned in this context are, for example, the solubility, the retrogradation behavior, the water-binding capacity, the film-forming properties, the viscosity, the pasting properties, the freeze-thaw stability, the stability to acids, the gel strength and the like.
The basic biochemical synthetic pathways which lead to the synthesis of starch are only roughly known. However, there exists a series of steps in which the detailed mechanisms which lead to the synthesis of the starch granules and of the starch are hitherto not elucidated and therefore still the subject of research.
It is currently not possible to influence the content of covalently bonded starch phosphate in plants by means of plant breeding alone.
An alternative to plant breeding methods is the targeted modification of starch-producing plants by recombinant methods. The prerequisite herefor, however, is the identification and characterization of the enzymes which participate in starch synthesis and/or in the modification of starch, and the isolation of the nucleic acid molecules which code for these enzymes and the subsequent functional analysis in transgenic plants.
In plant cells, starch synthesis takes place in the plastids, which are the chloroplasts in photosynthetically active tissue and the amyloplasts in photosynthetically inactive, starch-storing tissue. Important enzymes which play a role in starch synthesis are the R1 proteins (=alpha-glucan water dikinase, E.C. 2.7.9.4; Lorberth et al. (1998) Nature Biotechnology 16: 473-477), starch synthases and the branching enzymes (=BE; see, for example, Ponstein et al., Plant Physiol. 29 (1990), 234-241; Kossmann et al., 1991, Mol. Gen. Genet. 230, 39-44; Safford et al., 1998, Carbohydrate Polymers 35, 155-168; Jobling et al. 1999, The Plant Journal 18(2): 163-171). Branching enzymes catalyze the introduction of α-1,6-branchings into linear α-1,4-glucans. In the starch synthases, a variety of isoforms have been described, all of which catalyze a polymerization reaction by transferring a glucosyl residue from ADP-glucose to α-1,4-glucans.
An overview over native starches isolated from various plant species, where variations of enzymes which play a role in starch biosynthesis are observed, can be found in Kossmann and Lloyd (2000, Critical Reviews in Plant Sciences 19(3): 171-226).
Starch synthases (EC 2.4.1.21) can be divided into two classes: the starch-granule-bound starch synthases (“granule-bound starch synthases I”; GBSS I) and the soluble starch synthase (“soluble starch synthases”; SSS, also referred to as “SS”). This distinction is not unambiguous in each case since some of the starch synthases exist both in starch-granule-bound form and in soluble form (Denyer et al., Plant J. 4 (1993), 191-198; Mu et al., Plant J. 6 (1994), 151-159).
In contrast to the GBSSI, which leads to the synthesis of amylose, little is known as yet about the precise enzymatic function of the various classes of soluble starch synthase in starch biosynthesis.
The biochemical characterization resulted in the identification of soluble starch synthase proteins with molecular weights of between approximately 60 to approximately 180 kDa. The cloning of cDNAs which code for starch synthases made it possible to distinguish different classes which were defined as the result of sequence homologies and as the result of the functional characteristics of the (soluble) starch synthases.
To date, eight classes of starch synthases have been identified in higher plants (inter alia by Li et al. (2003) Funct. Intergr. Genomics 3:76-85):                starch-granule-bound starch synthase I (Granule-Bound Starch Synthase I=GBSS I) (rice: for example Okagaki (1992) Plant Mol. Biol. 19:513-516; potato: van der Leij et al. (1991) Mol. Gen. Genet. 228:240-248; maize: for example Kloesgen et al. (1986) Mol. Gen. Genet. 203:237-244);        soluble starch synthase I (=SSI; rice: Baba et al. (1993) Plant Physiol. 103:565-573; potato: Kossmann et al. (1999) Planta 208: 503-511; maize: Knight et al. (1998) Plant J. 14:613-622);        soluble starch synthase II (=SSII; pea: Dry et al. (1992) Plant J. 2:193-202, potato: Edwards et al. (1995) Plant J 8: 283-294, maize: Harn et al, (1998) Plant Mol. Biol. 37(4): 639-649; wheat: Walter et al. (1996) Genbank Acc. U66377; rice: Yamamoto and Sasaki (1997) Plant Mol. Biol. 35:135-144 and barley: Li et al. (2003), Funct. Integr. Genomics 3: 76-85);        soluble starch synthase III (=SSIII; potato: Abel et al. (1996) Plant J 10:981-991: fodder pea: GenBank Acc. No AJ225088);        soluble starch synthase IV (=SSIV; wheat: GenBank Acc. No AY044844);        soluble starch synthase V (=SSV; fodder pea: GenBank Acc. No VUN006752; Arabidopsis: GenBank Acc. No AL021713; maize: WO 97/26362) and        dull (Gao et al. (1998) Plant Cell 10:399-412;        soluble starch synthase VI (=SSVI; maize: WO 01/12826).        
The content of covalently bound starch phosphate varies, depending on the plant species. Thus, for example, certain maize mutants synthetize starch with an increased starch phosphate content (waxy maize 0.002% and high-amylose maize 0.013%), while traditional maize varieties only contain traces of starch phosphate. Small amounts of starch phosphate are also found in wheat (0.001%), while no starch phosphate was detected in oats and millet. Likewise, less starch phosphate was found in rice mutants (waxy rice 0.003%) than in traditional rice varieties (0.013%). Significant amounts of starch phosphate were detected in plants which synthetize tuber- or root-reserve starch such as, for example, tapioca (0.008%), sweet potato (0.011%), arrowroot (0.021%) or potato (0.089%). These percentages for the starch phosphate content are in each case based on the dry weight of starch and have been determined by Jane et al. (1996, Cereal Foods World 41 (11), 827-832). Studies on SSI-antisense potatoes revealed that their phosphate content was increased by 30-70% over the wild type (WO 96/15248).
WO 00/08184 describes plants in which both the activities of starch synthase III (=SSIII) and of branching enzyme I (=BEI) are reduced. In comparison with starch from wild-type plants, starch from such plants has an elevated phosphate content. Wheat plants which, as the result of the overexpression of an R1 gene from potato, have an increased activity of an R1 protein and an increased starch phosphate content are described in the international patent application WO 02/34923.
The distribution of phosphate in starch which has been synthesized by plants (native starch) is generally distinguished by the fact that approximately 30% to 40% of the phosphate residues are bonded covalently in the C3 position and approximately 60% to 70% of the phosphate residues in the C6 position of the glucose molecules (Blennow et al., 2000, Int. J. of Biological Macromolecules 27: 211-218). In contrast, chemically phosphorylated starches additionally have phosphate residues bonded in the C2 position of the glucose molecules since the chemical reaction proceeds in an undirected fashion.
WO 03/023024 discloses rice starches which have a DSC T-onset temperature of up to 69.5° C. and/or a DSC T-peak temperature of up to 73.6° C.; a total of approximately 400 rice varieties of the groups japonica and indica have been analyzed for these features.
Umemoto et al. (2002, Theor. Appl. Genet. 104:1-8) describe the analysis of back-crossed inbred lines between a japonica variety (Nipponbare) and an indica variety (Kasalath). They conclude from their results that the alk(t), gel(t) and acl(t) locus, which is responsible for the different gelatinization onset temperatures (DSC T-onset) between japonica and indica varieties, might be the starch synthase isoform SSIIa.
WO 03/023024 describes a rice transformant (#78-1) of the japonica variety (Kinmaze) into which a gene of starch synthase IIa (SSIIa) from the indica form IR36 has been transformed. The resulting changes in the amylopectin side-chain profile are shown and, as a consequence, indicate a shift from the japonica profile towards that of the indica variety (FIG. 22 in WO 03/023024).
WO 03/023024 describes neither phosphate contents, amylose contents nor rheological properties of the rice starches or flours.
The relationship between the change in the amylopectin side-chain profile, which is brought about by SSIIa, and the DSC T-onset temperature of the starches is again shown, in a recently published paper (Nakamura et al., (2005) PMB; 58(2): 213-27), with reference to the values for the transformants and the corresponding “recipient” and “donor” lines (FIG. 6B). FIG. 6B shows the temperatures for DSC T-onset of the starches of the SSIIa-transformants generated. In no case is the DSC T-onset higher than 70° C. The highest T-onset value detailed therein is 69.5° C., as described also in WO 03/023024 (pp. 21-24).
Thus, neither WO 03/023024 nor Nakamura et al. (2005) teach a way in which rice starches whose DSC T-onset temperature exceeds 70° C. can be generated.
However, a higher DSC T-onset temperature is desired insofar as it is an important feature of an improved thermal stability of starches and thus also for the change in the crystalline structure as the result of the effect of heat.